CN1160084C - Active centipede extract for suppressing cancer and its extracting process - Google Patents
Active centipede extract for suppressing cancer and its extracting process Download PDFInfo
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- CN1160084C CN1160084C CNB011002883A CN01100288A CN1160084C CN 1160084 C CN1160084 C CN 1160084C CN B011002883 A CNB011002883 A CN B011002883A CN 01100288 A CN01100288 A CN 01100288A CN 1160084 C CN1160084 C CN 1160084C
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Abstract
The present invention relates to an active cancer-suppressing centipede extractive and an extracting method thereof. In the extracting method, centipede coarse powder is degreased by alcohol to obtain residues extracted in water at a temperature of 4 to 8 DEG C and are frozen and dried after pressure reduction and concentration to obtain aqueous extractive freeze-drying powder; the freeze-drying powder is eluted by distilled water after column chromatography to collect color bands D, and the color bands D are frozen and dried after pressure reduction and concentration; the freeze-drying powder of the color bands D is decolorized by active carbon to obtain decolorized freeze-drying powder which is eluted by distilled water after column chromatography and is detected by UV220 for collecting a second peak (a main peak); and cancer-suppressing effective parts D-1 are obtained through rotary concentration at a temperature of 30 to 40 DEG C and freeze drying. The method has the advantages of simplicity, convenience and easy operation, and is favorable for researching and staring cancer-suppressing preparation using centipedes as main medicines.
Description
The present invention relates to the cancer-resisting substance of animal origin, relates in particular in animal Chinese medicine body and extracts active substance, more specifically to a kind of active centipede extract for suppressing cancer and extracting method thereof.
Extensively exist the large number of biological active substance in the animal Chinese medicine body, but the research to traditional animal Chinese medicine lags far behind plant amedica, and to the research of traditional animal Chinese medicine, particularly to the research of their activity in vivo compositions, be to safeguard and the enhancement human health, and, all can have very important significance by tool at the aspects such as basic law of illustrating some life sciences.
Scolopendra is China's traditional animal Chinese medicine commonly used, the key medicine that the town is painful in order to relieve dizziness, high fever, infantile convulsions, epilepsy, etc., dispersing pathogen accumulation, collateral dredging relieve the pain.Clinically be used for the treatment of spasm, inflammation, swell and ache etc., the treatment of tumor, hepatitis, nephritis is had many reports, for example, " clinical practice of Scolopendra " of Dong Hanliang, referring to the Hubei Journal of Traditional Chinese Medicine, 1982. (3) p26; " modern age Scolopendra Clinical advances " of Zhou Benhong, referring to Chinese medicine information, 1991. (8) 1 p45-48; With " Chinese traditional treatments of 62 routine primary lung cancers " of Shen Pian, referring to journal of shanghai Chinese medicine, 1982. (7) p9.These reports all are that Scolopendra is added in the prescription as Herba indigoferae Pseudotinctoriae, do not extract the active substance of Scolopendra.Very few to Scolopendra chemistry and active component research report both at home and abroad, though the biological activity to scorpion venom has some bibliographical informations in recent years, for example, " the Effects of anextract fromthe Centipede Scolopendra moristans on intestine; Uterus andheart contractions and on blod glucose and Liver and muscleglycogen Levels " of A.H Mohamed etc. is referring to Toxicon 18 (1,980 3) 581-589; " Proteins; Lipids; Lipoproteins and some enzymecharacteri-zations of the venom extract from the CentipedeScolopendra Morsitans " of A.H Mohamed etc. is referring to Toxicon 21 (3) 371-377; " the Biological activities of centipede crude venom " of Wang you etc. is referring to Kexuemongbao 1985.30 (8) 1102-1105; " chemical composition and the biological activity of Scolopendra (ScolopendraSubspinipes mutilans L.Koch) poison " with Wu Gang etc., referring to journal of biological chemistry 1992.8 (2) 144-149, do not have the tumor-suppression activity composition but all clearly mention in these reports from the water soluble ingredient of Scolopendpa Subspinipes Mutilans L. KOCH complexity, seeking, particularly belong to the chemical compound composition of peptide class character.For example, " separation and purification of Scolopendra basic protein SSMP-d and the evaluation of part physicochemical property thereof " that Xu Ming ticks etc., referring to journal of biological chemistry 1997.13 (5) 586-591, it is from Scolopendpa Subspinipes Mutilans L. KOCH water-soluble alkaline protein part, obtains having the active component of obvious inhibition KB (human oral cavity epithelial scale cancer) and HCT (human colon cancer cell) human cancer cell.Therefrom separation, purification, identified that molecular weight is that polypeptide SSM-peptide-1 and the molecular weight of 5.8kd is that 24.64kd, isoelectric point, IP are 9.27 protein S SMP-d, and measured the partial amino-acid series of the N-end of SSMP-d, but it is a Scolopendra water-soluble macromolecule albumen, and tumor-suppression activity can not show a candle to extract D-1 of the present invention.Therefore be necessary very to grope that a whole set of separates targetedly, purification process and accordingly to research, the development technique of animal Chinese medicine, obtain to have the live part of tumor-suppression activity in the Scolopendpa Subspinipes Mutilans L. KOCH body, and it has been carried out more deep chemistry and pharmacological research.
The purpose of this invention is to provide a kind of Scolopendra activity extract, this extract is the water miscible cancer effective ingredient that presses down, thereby avoided being used for Anti-cancer Chinese medicine formula as Chinese medicine simply with Scolopendra, so both can directly extract of the present invention be used for anticancer prescription, also it can be used for anticancer compound preparation as principal agent.
Another object of the present invention provides a kind of method of extracting the water-soluble anticancer active matter in the Scolopendra body, thereby obtains to have the composition of tumor-suppression activity in the Scolopendra body.
The invention provides a kind of active centipede extract for suppressing cancer, described extract shows below physicochemical characteristic:
1) outward appearance: colourless lyophilized powder;
2) dissolubility: very easily water-soluble;
3) molecular weight: 500-2000;
4) the main composition: lysine, glutamic acid, serine, glycine, alanine, aspartic acid, proline etc.;
5) elementary analysis data: C:27-28%, H:7-8%, N:13-15%;
6) UV scanning collection of illustrative plates: there is absorption maximum at the 220nm place; And described extract is to adopt following method to extract:
1) Scolopendra is worn into coarse powder, get coarse powder after the alcohol extraction defat, carry at 4-8 ℃ of water residue obtained, concentrating under reduced pressure again, lyophilizing gets lyophilized powder;
2) with described lyophilized powder through the column chromatography first time, the distilled water eluting is collected colour band D, with lyophilizing behind the D part concentrating under reduced pressure, D part lyophilized powder;
3) with D lyophilized powder lyophilizing behind decolorizing with activated carbon partly, the lyophilized powder that must decolour, again through the column chromatography second time, the distilled water eluting, UV220 detects, and collects second peak (main peak), and 30-40 ℃ of concentrated, the lyophilizing of rotation promptly gets the D-1 extract.
Extracting method of the present invention is achieved in that 1) Scolopendra is worn into coarse powder, get coarse powder through alcohol degreasing, carry at 4-8 ℃ of water residue obtained, lyophilizing gets the water extract lyophilized powder behind the concentrating under reduced pressure again; 2) described lyophilized powder is through the column chromatography first time, and the distilled water eluting is collected colour band D, with lyophilizing behind the D part concentrating under reduced pressure, gets D part lyophilized powder; 3) with D lyophilized powder lyophilizing behind decolorizing with activated carbon partly, the lyophilized powder that must decolour, again through the column chromatography second time, the distilled water eluting, UV220 detects, and collects second peak (main peak), and 30-40 ℃ of concentrated, the lyophilizing of rotation promptly gets D-1 extract of the present invention.
Term used herein " Scolopendra " is meant Scolopendpa Subspinipes Mutilans L. KOCH.
The present invention be have in the Anti-cancer Chinese medicine formula according to clinical practice more Scolopendra be main flavour of a drug this truely be basis, after comprehensive screening to the different extracts of Scolopendra, carried out separation, purification to having the active part of anticancer, obtained the laboratory animal inside and outside and all have the obviously live part D-1 of cancer suppressing action.Its molecular weight ranges is about 500-2000.Scolopendra water-soluble alkali albumen is different materials in extract of the present invention and the prior art.The of the present invention searching out from the water soluble ingredient of Scolopendpa Subspinipes Mutilans L. KOCH complexity has the tumor-suppression activity composition, particularly belongs to the chemical compound composition of peptide class character, and this still belongs to the first.To obtain have the tumor-suppression activity composition in the Scolopendra body, need grope that a whole set of separates targetedly, purification process and accordingly to research, the development technique of animal Chinese medicine.Process of the present invention is simple and easy to do, and purposiveness is strong, helps development and utilization from now on.
Fig. 1 is the process chart of the inventive method.
Fig. 2 is the UV scanning collection of illustrative plates of extract D-1 of the present invention.
Fig. 3 is the high-pressure liquid phase collection of illustrative plates of the extract D-1 of one embodiment of the invention.
Fig. 4 is the high-pressure liquid phase collection of illustrative plates of the extract D-1 of another embodiment of the invention.
Fig. 5 is the high-pressure liquid phase collection of illustrative plates of the extract D-1 of another embodiment of the invention.
According to one embodiment of the invention, 1) with Scolopendpa Subspinipes Mutilans L. KOCH slightly behind alcohol degreasing, carry 4-8 ℃ of water at low temperature, water extract lyophilizing behind rotation concentrating instrument concentrating under reduced pressure gets lyophilized powder, and the weight of its lyophilized powder is about 10% of coarse powder, 2) lyophilized powder is through the column chromatography first time, the distilled water eluting is collected colour band D, with lyophilizing behind the D part eluent concentrating under reduced pressure, get the lyophilized powder of D part, 3) D part is behind activity carbon column decoloring, again through the column chromatography second time, and the distilled water eluting, collect second peak (main peak), 30-40 ℃ of low temperature rotation concentrates, lyophilizing promptly gets extract D-1, is about 0.1% left and right sides (see figure 1) of coarse powder amount.Extract obtained have a following physico-chemical property:
1) outward appearance: colourless lyophilized powder;
2) dissolubility: very easily water-soluble;
3) molecular weight: 500-2000;
4) the main composition: lysine, glutamic acid, serine, glycine, alanine, aspartic acid, proline etc.;
5) elementary analysis data: C:27-28%, H:7-8%, N:13-15%;
6) UV scanning collection of illustrative plates: there is the absorption maximum (see figure 2) at the 220nm place
The ethanol that is used for step 1) of the present invention takes off ester and adopts conventional method to carry out, and for example takes off ester with 95% ethanol.
Be used for step 2 of the present invention) the first time column chromatography preferably available from the glucose gel Sephadex G-25 of Sweden Pharmacia, wherein the ratio of gel filtration chromatography sample and gel is 1: 100-120, more excellent is 1: 100.
Be used for step 3) of the present invention the second time column chromatography preferably available from the glucose gel Sephadex G-15 of Sweden Pharmacia, wherein the ratio of gel filtration chromatography sample and gel is 1: 80-100, more excellent is 1: 80.
Water at low temperature temperature raising degree of the present invention is preferably 4-8 ℃, and more excellent is 4 ℃.The temperature of concentrate under reduced pressure at low temperature of the present invention is preferably 30-40 ℃, and more excellent is 30 ℃, and concentrating under reduced pressure is to adopt common process to carry out.
The activated carbon that is used for step 3) of the present invention preferably uses the GH-15 activated carbon of brilliance timber mill, Beijing, and wherein the ratio of decolorizing with activated carbon post sample and activated carbon is about 1: 16.
According to another embodiment of the invention, the active anticancer of extract is tested.
Result of the test shows: no matter be to adopt colony forming method or mtt assay to detect, macromole A part is to the IC of KB cell (human oral cavity epithelial scale cancer)
50At 10-25 μ g/ml; Micromolecule D part is to KB, HCT (human colon carcinoma), Bel
7402 (People's hepatocarcinoma) and A
2780The IC of (human ovarian cancer) cell
50Be respectively 19.5,19.7,20.6 and 13.7 μ g/ml; The D part is decoloured through further, means such as separation and purification, and the micromolecular compound D-1 component that obtains is at KB, HCT, Bel
7402And A
2780The IC of cell
50Be respectively 3.7,8.4,6.6 and 1.2 μ g/ml, this test is through repeatedly repeating, as a result unanimity.Therefore, think Scolopendpa Subspinipes Mutilans L. KOCH coarse powder water extract, after separation and purification, macromole A part and micromolecule D part all have certain active anticancer to many strains human cancer cell.Through after being further purified, the D-1 component that obtains is stronger than the activity of D part again, and its activity can improve 3-10 doubly.
Give mouse inoculation transplanted tumor, hepatocarcinoma H
22And behind the Lewis lung cancer, with continuous lumbar injection 9-10 of D-180-160mg/kg/ day days, its suppression ratio can reach 25-56%, and (P＜0.05-0.01) illustrate that D-1 injects in vivo tangible cancer suppressing action is also arranged.
Below with reference to following indefiniteness embodiment the present invention is described in more details.
Scolopendpa Subspinipes Mutilans L. KOCH coarse powder 600 grams, behind 95% alcohol degreasing, 4 ℃ of water of low temperature are carried, and lyophilizing behind 30 ℃ of concentrating under reduced pressure of water extract low temperature gets lyophilized powder 70 grams.Lyophilized powder is through Sephadex G-25 column chromatography, and the distilled water eluting is collected colour band D, with lyophilizing behind the D part concentrating under reduced pressure, gets lyophilized powder 16 grams.The D part is behind decolorizing with activated carbon, and lyophilized powder 2.6 grams must decolour.Again through Sephadex G-15 column chromatography, the distilled water eluting, UV220 detects, and collects second peak (main peak), and concentrating under reduced pressure, lyophilizing promptly get D-1.Must measure is 634 milligrams.Its HPLC (high performance liquid chromatography) chromatography collection of illustrative plates as shown in Figure 2.Wherein HPLC chromatography technology is: post: C
18(φ 4.6 * 250mm) for 300A; Eluent: 0.1% TFA; Ultraviolet detection: 220nm (ultraviolet 220nm).Uv absorption shows 6 peaks, and wherein retention time is to locate about 6 minutes to be main peak, and content is about 50%.
Scolopendpa Subspinipes Mutilans L. KOCH coarse powder 600 grams, behind 95% alcohol degreasing, 8 ℃ of water of low temperature are carried, and lyophilizing behind 40 ℃ of concentrating under reduced pressure of water extract low temperature gets lyophilized powder 60 grams.Lyophilized powder is through Sephadex G-25 column chromatography, and the distilled water eluting is collected colour band D, with freeze behind the D part concentrating under reduced pressure in, lyophilized powder 15 grams, the D part is behind decolorizing with activated carbon, lyophilized powder 2.5 grams must decolour.Through Sephadex G-15 column chromatography, carry out the HPLC chromatography with the method for embodiment 1 again, UV220 detects (ultraviolet 220), and the distilled water eluting is collected second peak (main peak), and concentrating under reduced pressure, lyophilizing promptly get D-1.Must measure is 587 milligrams.Its HPLC (high performance liquid chromatography) chromatography collection of illustrative plates as shown in Figure 3.
Embodiment 3
Scolopendpa Subspinipes Mutilans L. KOCH coarse powder 600 grams, behind 95% alcohol degreasing, 6 ℃ of water of low temperature are carried, and lyophilizing behind 30 ℃ of concentrating under reduced pressure of water extract low temperature gets lyophilized powder 60 grams.Lyophilized powder is through Sephadex G-25 column chromatography, and the distilled water eluting is collected colour band D, with lyophilizing behind the D part concentrating under reduced pressure, gets lyophilized powder 15 grams, and the D part is behind decolorizing with activated carbon, and lyophilized powder 2.5 grams must decolour.Through Sephadex G-15 column chromatography, carry out the HPLC chromatography with the method for embodiment 1 again, UV220 detects, and the distilled water eluting is collected second peak (main peak), and concentrating under reduced pressure, lyophilizing promptly get D-1.Must measure is 610 milligrams.Its HPLC (high performance liquid chromatography) chromatography collection of illustrative plates as shown in Figure 3.
Scolopendra extract of the present invention is carried out following test:
In vitro tests-
1. mtt assay: with the human cancer cell of various exponential phases, be inoculated in 96 orifice plates, every hole 800-1500 cell, dosing after 24 hours is at RPMI1640 (GIBCO) (containing 10% calf serum) culture medium, 37 ℃, 5%CO
2Cultivated under the condition 4 days, and added MTT, with Bole's 450 microplate reader, 450 and the 570nm dual wavelength detect, calculate inhibitory rate of cell growth, and obtain IC
50
2. colony forming method: with the human cancer cell of various exponential phases, be inoculated in the culture dish of diameter 6cm, 50 cell/ml, every blood 5ml, dosing after 6 hours is at RPMI1640 culture medium, 37 ℃, 5%CO
2Cultivate under the condition after 8 days, with Giemsa dyeing, counting colony (50 cell/colonies) calculates suppression ratio and IC
50
Experiment in the body-
Get well-grown mice transplanted tumor Lewis lung cancer and hepatocarcinoma H
22, learn requirement by anticarcinogen drug effect word, routine is inoculated in mice.In inoculation grouping in 24 hours administration, every group of 10 mices are established matched group and but the various dose group of cancer live part D-1 simultaneously.Injection on an empty stomach, 9-10 days continuously, put to death animal in 24 hours after the drug withdrawal, the stripping tumor is weighed and is calculated tumour inhibiting rate, carries out statistics t check deal with data.
More than used material be:
The KB of cell strain-1.: the human oral cavity epithelial squamous cell carcinoma is Bel 2.
7402: human liver cancer cell
3. HCT: human colon cancer cell is A 4.
2780: Proliferation of Human Ovarian Cell
Transplanted tumor-rat liver cancer H
22And LeWis pulmonary carcinoma.
Above tumor strain is this laboratory and preserves.
Laboratory animal-employing Kunming and pure lines C
57/ BL mice, body weight 18-23 gram is provided by Chinese Academy of Medical Sciences's animal center, and the quality certification number (Kunming mouse: 01-3001, C send out in Beijing animal committee
57/ BL:01-3004.
Concrete experimental result is as follows:
1, in the Scolopendpa Subspinipes Mutilans L. KOCH each component to the cytotoxicity of human cancer cell
From isolated each component of Scolopendpa Subspinipes Mutilans L. KOCH water-solubility protein part, no matter be to detect with colony forming method or mtt assay, macromole A component is to the IC of kB cell
50All at 10-25 μ g/ml; Micromolecule D component is to KB, HCT, Bel
7402And A
2780IC
50Be respectively 119.5,19.7,20.6 and 13.7 μ g, repeated experiments result is approximate; The D component obtains D-1 through nearly single step purification again, at KB, HCT, Bel
7402And A
2780The IC of human cancer cell strain
50, be respectively 3.7,8.4,6.6 and 1.2 μ g/ml (seeing Table-1).D-1 good water solubility, physicochemical property are stablized, cytotoxic activity obviously improves, and are worth entering the active observation of vivo antitumor.
Each component of table-1 Scolopendpa Subspinipes Mutilans L. KOCH is to the cytotoxicity of human cancer cell strain
| Component | IC 50(μg/ml) | |||
| KB | HCT | Bel 7402 | A 2780 | |
| A D D-1 | 10-25 19.5 19.6 3.7 | 19.7 21.1 8.4 | 20.6 22.8 6.4 | 13.7 1.2 |
2, D-1 observes the antitumaous effect of transplanted tumor
1. to rat liver cancer H
22Clinical trial: mice is given mice i.P D-1 100 or 150mg/kg in back 24 hours of inoculation, successive administration 10 days, result and matched group comparison, its tumor control rate is respectively 32% and 25% (P＜0.05) (seeing Table-2).
Table-2 D-1 are to rat liver cancer H
22Observation of curative effect
| Group | Dosage (mg/kg) | Number of animals (beginning/end) | Body weight (g) | Tumor heavy (g) | Suppression ratio (%) | The P value |
| Contrast D-1 | / 100×10 | 10/10 10/10 | 19.8/+6.1 19.9/+4.6 | 2.45±0.85 1.66±1.11 | / 32.0 | ＜0.05 |
| Contrast D-1 | / 150×10 | 10/10 10/10 | 20.1/+7.1 19.9/+5.6 | 4.06±1.05 3.05±0.69 | / 25.0 | ＜0.05 |
2. to the observation of curative effect of Mice Bearing Lewis Lung Cancer: mice was given mice i.pD-1 150mg/kg or 80,120 and 160mg/kg in back 24 hours in inoculation, successive administration 9 days, result and matched group compare, and its tumor kind suppression ratio is respectively 49.1% and 50.2% (P＜0.01), 39.9% (P＜0.05), 55.5% (P＜0.01).See Table-3
Table-3 D-1 are to the observation of curative effect of Mice Bearing Lewis Lung Cancer
| Group | Dosage (mg/kg) | Number of animals (beginning/end) | Body weight (beginning/end) | Tumor heavy (g) | Suppression ratio (%) | The P value |
| Contrast D-1 | / 150×9 | 10/10 10/10 | 18.9/+1.4 20.3/+0.5 | 2.67±0.57 1.36±0.85 | / 49.1 | / ＜0.05 |
| Contrast D-1 | / 80×9 120×9 160×9 | 10/10 10/10 10/10 10/10 | 22.0/+1.5 21.1/+3.7 22.1/+2.8 22.6/+2.3 | 2.8±1.10 1.40±0.42 1.69±0.92 1.25±0.70 | / 50.2 39.9 55.5 | / ＜0.01 ＜0.05 ＜0.01 |
Above Preliminary experiment results shows that the D-1 component that obtains from the separation of Scolopendpa Subspinipes Mutilans L. KOCH water-soluble portion is obvious to the cytotoxicity of several human cancer cells.Press down in the tumor experiment, in integral body rat liver cancer H
2And the antitumaous effect of Lewis lung cancer also obtains satisfied confirmation.
Though the present invention is described in detail particular,, those skilled in the art can understand that these technical schemes just illustrate and are not construed as limiting, scope of the invention claims limit.
Claims (16)
1, active centipede extract for suppressing cancer is characterized in that described extract adopts following method to extract:
1) Scolopendpa Subspinipes Mutilans L. KOCH is worn into coarse powder, get coarse powder through alcohol degreasing, carry at 4-8 ℃ of water residue obtained, lyophilizing gets the water extract lyophilized powder behind the concentrating under reduced pressure again;
2) described lyophilized powder is through the Sephadex G-25 column chromatography first time, and the distilled water eluting is collected colour band D, with lyophilizing behind the D part concentrating under reduced pressure, gets D part lyophilized powder;
3) with D lyophilized powder lyophilizing behind decolorizing with activated carbon partly, lyophilized powder must decolour, again through the Sephadex G-25 column chromatography second time, the distilled water eluting, UV220 detects, and collects second peak (main peak), 30-40 ℃ of concentrated, the lyophilizing of rotation, promptly get the D-1 extract, this extract shows below physicochemical characteristic:
1) outward appearance: multicolored lyophilized powder;
2) dissolubility: very easily water-soluble:
3) molecular weight: 500-2000;
4) the main composition: lysine, glutamic acid, serine, glycine, alanine, aspartic acid, proline etc.;
5) analytical data: C:27-28%, H:7-8%, N:13-15%:
6) UV scanning collection of illustrative plates: there is absorption maximum at the 220nm place.
2, extract as claimed in claim 1 is characterized in that the water temperature raising degree of described step 1) is 4 ℃.
3, extract as claimed in claim 1 is characterized in that described first time, column chromatography was glucose gel Sephadex G-25 column chromatography, wherein lyophilized powder: gel=1: 100-120.
4, extract as claimed in claim 3 is characterized in that the lyophilized powder of described Sephadex G-25 column chromatography: gel=1: 100.
5, extract as claimed in claim 1 is characterized in that described second time, column chromatography was glucose gel Sephadex G-15 column chromatography, wherein lyophilized powder: gel=1: 80-100.
6, extract as claimed in claim 5 is characterized in that the lyophilized powder of described SephadexG-15 column chromatography: gel 21: 80.
7, extract as claimed in claim 1 is characterized in that in the described decolorizing with activated carbon process D part lyophilized powder: activated carbon=1: 16.
8, extract as claimed in claim 1 is characterized in that described low temperature rotation thickening temperature is 30 ℃.
9, a kind of method of extracting the tumor-suppression activity thing from Scolopendra is characterized in that said method comprising the steps of:
1) Scolopendpa Subspinipes Mutilans L. KOCH is worn into coarse powder, gets coarse powder through the alcohol extraction defat, carries at 4-8 ℃ of water residue obtained, and lyophilizing gets the water extract lyophilized powder behind the concentrating under reduced pressure again;
2) described lyophilized powder is through the Sephadex G-25 column chromatography first time, and the distilled water eluting is collected colour band D, with lyophilizing behind the D part concentrating under reduced pressure, gets D part lyophilized powder:
3) with D lyophilized powder lyophilizing behind decolorizing with activated carbon partly, the lyophilized powder that must decolour, again through the Sephadex G-25 column chromatography second time, the distilled water eluting, UV220 detects, and collects second peak (main peak), and 30-40 ℃ of concentrated, the lyophilizing of rotation promptly gets the D-1 extract.
10, method as claimed in claim 9 is characterized in that the water temperature raising degree of described step 1) is 4 ℃.
11, method as claimed in claim 9 is characterized in that described first time, column chromatography was that glucose gel S is hadex G-25 column chromatography, wherein lyophilized powder: gel=1: 100-120.
12, method as claimed in claim 11 is characterized in that the lyophilized powder of described SephadexG-25 column chromatography: gel=1: 100.
13, method as claimed in claim 9 is characterized in that described second time, column chromatography was glucose gel Sephadex G-15 column chromatography, wherein lyophilized powder: gel=1: 80-100.
14, method as claimed in claim 13 is characterized in that the lyophilized powder of described Sephadex G-15 column chromatography: gel=1: 80.
15, extract as claimed in claim 11 is characterized in that in the described decolorizing with activated carbon process D part lyophilized powder: activated carbon=1: 16.
16, extract as claimed in claim 1 is characterized in that described low temperature rotation thickening temperature is 30 ℃.
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| CN102133233B (en) * | 2011-03-03 | 2013-08-14 | 湖南中医药大学 | Centipede extract capable of resisting tumor activity and preparation method thereof |
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